Reactivity of Porcine Epidemic Diarrhea Virus Structural Proteins to Antibodies against Porcine Enteric Coronaviruses: Diagnostic Implications

(i) Experimental design.The study was conducted with the approval of the Iowa State University Office for Responsible Research. Seven-week-old pigs (n = 72) were purchased from a conventional wean-to-finish farm with no history of porcine coronavirus infections. The pigs were prescreened for evidence of infection with PEDV, TGEV, PRCV, and PDCoV. Pig fecal swabs were tested by a PEDV N gene-based reverse transcription-PCR (rRT-PCR) (21) and PDCoV M gene-based rRT-PCR (14), while pig fecal and nasal swabs were tested by TGEV (S gene)/PRCV (N gene)-based differential rRT-PCR (20). The pigs' serum samples were tested with a PEDV immunofluorescence assay (IFA) (21), PEDV WV ELISA (24), TGEV/PRCV differential ELISA (Svanova, Sweden), and PDCoV IFA (14). Animals (n = 72) were randomized into six groups; each group consisted of 12 pigs in one room, with 6 pens per room and 2 pigs per pen. Details related to virus strains and the routes of experimental inoculation are presented in Table S1 in the supplemental material. The age-related infectious dose for each virus was previously determined in a pilot study (data not shown). The pigs were closely observed twice daily for clinical signs throughout the study. A total of 792 serum samples were collected from each group on days postinfection (DPI) −7, 0, 3, 7, 10, 14, 17, 21, 28, 35, and 42. Virus shedding within groups and absence of cross-contamination between groups during the observation period (−7 to 42 DPI) were confirmed by rRT-PCR, whereas seroconversion within inoculation groups was confirmed by ELISA or IFA (data not shown). At 42 DPI, all pigs were humanely euthanized by the use of a penetrating captive bolt (Accles and Shelvoke, Ltd., Sutton Coldfield, United Kingdom) followed by exsanguination.

Five different polypeptides corresponding to the four PEDV structural proteins (S, N, M, and E) were recombinantly generated; PEDV WV particles were purified from cell culture by ultracentrifugation, as described elsewhere (24). The PEDV recombinant and WV target antigens were used to develop a multiplex (6-plex) FMIA platform to analyze the antibody response to each target using sera from pigs experimentally inoculated with PEDV, TGEV Miller and Purdue strains, PRCV, PDCoV, or a sham control. The diagnostic sensitivity and specificity and the analytical specificity were evaluated for each individual antigen.

(ii) Generation of PEDV recombinant spike (rS)-derived proteins.The coding regions of the amino-terminal receptor-binding (S1) domains derived from a consensus sequence (Fig. 4) based on 10 PEDV non-S-INDEL strains (2,151 nucleotides [nt]) or 5 S-INDEL strains (2,142 nt) (see Table S2 in the supplemental material) were codon optimized for expression in mammalian cells and synthetically produced (Shanghai Genery Biotech Co., Ltd.) with the addition of a 5′ terminal eukaryotic native signal (encoding MKSLTYFWLFLPVLSTLSLP) and a 3′ terminal tobacco etch virus (TEV) cysteine protease site (encoding ENLYFQS), followed by the Fc portion of human IgG1 (GenBank accession number JX292764.2 ). The genes were amplified or manipulated using forward primers F1-(5′-TAA ACG GAT CTC TAG CGA ATT CGC CGC CAC CAT GAA GAG CCTG-3′) and F2-(5′-CTT CCA GAG CGG CTC CGA CAA GAC CCA CAC CGT CGA GTG CCC ACC GTG CCC AG-3′) and reverse primers R1-(5′-CGA GCG GCC GCT AGA AGC TTT CAT TTA CCC GGA GAC AGG GAG-3′) and R2-(5′-GTC GGA GCC GCT CTG GAA GTA CAG GTT CTC GTG ATA GAA GAA TCC GGG CAG-3′). Amplicons were cloned into pNPM5 eukaryotic expression vector (Novoprotein, Short Hills, NJ, USA), and the recombinant plasmids were transiently transfected into human embryonic kidney 293 (HEK293) cells (Invitrogen, Thermo Fisher Scientific, Grand Island, NY, USA) (1 × 10⁶ cells/ml) using polyethylenimine (PEI) (Thermo Fisher Scientific) at an optimal 1:4 (wt/wt) plasmid/PEI ratio. Transfected HEK293 cells were grown in serum-free FreeStyle 293 expression medium (Gibco, Life Technologies, Carlsbad, CA, USA) at 37°C with 5% CO₂ by orbital shaking at 120 rpm. At day 5 posttransfection, culture supernatants were harvested by centrifugation at 3,500 × g for 20 min and were subjected to filter sterilization using a 0.45-μm-pore-size filter. The soluble expression of Fc tag-fused S1 proteins (S1-Fc) (107.5 kDa) was confirmed by 12% dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). PEDV S1-Fc proteins were enzymatically cleaved by incubation with TEV (20 IU/mg sample) for 3 h at 25°C under conditions of endotoxin control and purified from culture supernatant by protein A chromatography (GE Healthcare, Pittsburgh, PA, USA) and nickel (Ni)-chelating Sepharose Fast Flow (SFF) affinity chromatography (GE Healthcare), according to the manufacturer's instructions. Purified rS1 non-S-INDEL (717-amino-acid [aa]) and rS1 S-INDEL (714-aa) proteins were dialyzed against phosphate-buffered saline (PBS) (10 mM phosphate and 150 mM NaCl, pH 7.4) and analyzed by SDS-PAGE and Western blotting.

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FIG 4

Amino acid sequences of 5 recombinant polypeptides derived from the following PEDV structural proteins: (i) envelope (E) full-length protein (CV777 PEDV strain); (ii) full-length nucleocapsid (N) protein from a consensus among PEDV strains (CV777, non-S-INDEL, and S-INDEL); (iii) truncated (C-terminal intravirion topological domain) membrane (M) protein from a consensus among PEDV strains (CV777, S-INDEL, and non-S-INDEL); (iv) truncated spike protein (globular head; S1) from a consensus among PEDV non-S-INDEL strains; (v) S1 from a consensus among PEDV S-INDEL strains.

(iii) Generation of PEDV recombinant nucleocapsid (rN) protein.An Escherichia coli codon-optimized consensus version (Fig. 4) obtained from a multiple-sequence alignment (non-S-INDEL, S-INDEL, and CV777 strains) of the full-length PEDV N (1,356-nt) gene (see Table S2 in the supplemental material) was synthesized in vitro (Shanghai Genery Biotech Co., Ltd., Shanghai, China). The gene was amplified using forward primer 5′-CAT CAT CAT CAT CAT CAT ATG GCA TCT GTT AGC TTT CAG GAT CG-3′ and reverse primer 5′-AGA CTG CAG GTC GAC AAG CTT TTA ATT GCC GGT ATC GAA GAT C-3′. The amplicon was cloned into pCold II expression plasmid (Novoprotein Scientific Inc., Shanghai, China), confirmed by sequencing (Genewiz Inc., Suzhou, China), and then transformed into an E. coli BL21(DE3) host strain (Invitrogen, Carlsbad, CA, USA). The transformants were resuspended and grown in 1 liter of Luria-Bertani (LB) medium (Invitrogen) containing 100 μg/ml ampicillin at 16°C with shaking at 250 rpm. When an A₆₀₀ value of 0.9 was reached, 0.1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was added, and cultures were grown for an additional 16 h at 16°C. Cells were chilled at 4°C and harvested by centrifugation at 3,500 × g for 15 min, resuspended in 150 ml of lysis buffer (20 mM Tris-HCl and 500 mM NaCl, pH 8.0), and disrupted by ultrasonication (Vibra-Cell sonicator; Sonics & Materials, Newtown, CT, USA). The crude extracts were centrifuged at 15,000 × g for 30 min at 4°C, and the soluble expression of a histidine (His) tag-fused N-PEDV protein (50.4 kDa) was confirmed by SDS-PAGE analysis. The rN protein (452 aa) was purified from the soluble fraction by Ni-chelating SFF affinity chromatography (GE Healthcare) according to the manufacturer's instructions. After separation of thrombin cleavage products on the Ni²⁺ column, HiTrap Phenyl High Performance (HP) (GE Healthcare) hydrophobic interaction chromatography and HiTrap SulfoPropyl (SP) HP strong cation exchange chromatography (GE Healthcare) were consecutively applied according to the manufacturer's instructions. Protein elutions were pooled, dialyzed against PBS (pH 7.4) at 4°C, and analyzed by SDS-PAGE and Western blotting.

(iv) Generation of PEDV recombinant membrane (rM) protein.An E. coli codon-optimized consensus version (Fig. 4) from multiple alignment (non-S-INDEL, S-INDEL, and CV777 strains) of the PEDV M (429-nt) gene encoding a truncated fragment corresponding to the C-terminal intravirion topological domain of the M protein (see Table S2 in the supplemental material) was synthetically generated (Shanghai Genery Biotech Co., Ltd.). The gene was amplified using forward primer 5′-CAT CAT CAT CAT CAT CAT ATG TTT GTG AAT AGT ATT CGC TTA TGG-3′ and reverse primer 5′-AGA CTG CAG GTC GAC AAG CTT TTA AAC CAG ATG CAG AAC TTT TTC G-3′. PCR products were cloned, sequenced, and transformed into BL21(DE3) cells as described above for the N protein. The truncated His-tagged fusion M-PEDV polypeptide (15.7 kDa; 143 aa), expressed in the precipitate of cell lysate as an inclusion body, was solubilized using a denaturing buffer (20 mM Tris-HCl, 300 mM NaCl, 8 M urea, and 5 mM β-mercaptoethanol; pH 8.0) and purified by Ni-chelating chromatography (GE Healthcare). Elutions were pooled, dialyzed against 20 mM phosphate buffer–150 mM NaCl–6 M urea (pH 7.4) at 4°C, and analyzed by SDS-PAGE and Western blotting.

(v) Generation of recombinant envelope (rE) small membrane protein.A commercial E. coli-expressed PEDV (strain CV777) recombinant His-tagged E protein (76 aa) was purchased from Cusabio (CSB-EP771125PPW; Cusabio Biotech Co., MD, USA), and its sequence is presented in Fig. 4.

(vi) PEDV multiplex fluorescent microbead-based immunoassay (FMIA).The covalent coupling of purified PEDV WV antigen and recombinant polypeptides to microbead sets was performed as previously described (43). A total of 25 μg of each protein, i.e., rS1 non-S-INDEL (0.6 mg/ml; bead region 53), rS1 S-INDEL (0.3 mg/ml; bead region 45), rN (0.44 mg/ml; bead region 64), rM (1 mg/ml; bead region 12), and rE (1 mg/ml; bead region 54), and 60 μl of purified WV antigen were coupled to 5 × 10⁶ carboxylated magnetic microspheres (MagPlex-C Microspheres; Luminex Corp., Austin, TX, USA). Each set of coupled beads was individually evaluated using different assay buffers to exclude matrix inhibitory effects and to select the optimum buffer combination for all bead sets. After it was verified that each of the bead sets worked properly in a single-analyte assay, they were sequentially added into progressively larger multiplex assays to exclude interference between bead regions. Serum samples were diluted to 1/50 in assay buffer (Sea Block Blocking Buffer; Thermo Fisher Scientific) and mixed with 50 μl of the bead suspension (∼2,500 beads per well for each target) in each well (Bio-Plex Pro flat-bottom plates; Bio-Rad Laboratories Inc., Hercules, CA, USA). The plates were incubated for 30 min on a microplate shaker (VWR, Radnor, PA, USA) at 400 rpm and were washed three times with 200 μl of 0.1 M PBS (pH 7.4) containing 0.1% Tween 20 (PBST). All incubations were performed at ∼22°C in a dark environment. Next, 50 μl of biotin-labeled goat anti-pig IgG (Fc) (Bethyl Laboratories Inc., Montgomery, TX, USA) at a 1/3,000 dilution in assay buffer was added to each well, followed by a 30-min incubation. After a washing step, 50 μl streptavidin phycoerythrin (SAPE; Moss Inc., Pasadena, MD, USA) at a 1/100 dilution in assay buffer was added to each well and the plates were incubated for 30 min. After an additional wash step, the microspheres were resuspended in 100 μl of assay buffer and analyzed using a dual-laser Bio-Plex 200 instrument (Bio-Rad Laboratories, Inc.). The events were gated to exclude doubles and other aggregates (Bio-Plex Manager software 6.0; Bio-Rad Laboratories, Inc.). The antibody response, reported as median fluorescent intensity (MFI), was expressed as the sample/positive result (S/P) ratio as follows: S/P ratio=(sample FI−background control mean FI)(positive control mean FI−background mean FI)

(vii) Data analysis.The cutoff value for and diagnostic performance of each individual PEDV antigen were determined by receiver operating characteristic (ROC) analysis (SAS version 9.4; SAS Institute, Inc., Cary, NC, USA) using 6-plex FMIA and/or WV ELISA results. Porcine coronavirus-negative samples (n = 252) were used to estimate diagnostic specificity, and PEDV-positive samples (n = 72) collected from the 12 PEDV-inoculated pigs between DPI 14 and 42 were used to estimate diagnostic sensitivity, time of detection, and over-time detection through the observational period. The analytical specificity (cross-reactivity) of each PEDV antigen was evaluated using samples (n = 384) collected between DPI 7 and 42 from animals inoculated with TGEV Miller and Purdue strains, PRCV, and PDCoV.

Pearson's chi-square test was used to detect differences in the numbers of seropositive animals over time (by DPI) among the individual antigens. Fisher's exact test was used when 25% of the cells in a contingency table contained counts of <5 (SAS version 9.4; SAS Institute, Inc., Cary, NC, USA). One-way analysis of variance (ANOVA) with Tukey's correction was used for multiple comparisons with an alpha value of 0.05 (GraphPad Prism 6). Specifically, we compared differences in the antibody responses of the inoculation groups by day postinoculation for each antigen target.